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A novel biased antagonist of the eotaxin-CCR3 pathway in eosinophils
Abstracts AB163
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
A Novel Protease, PRSS33 (serine protease 33), Is Specifically and Constitutively Expressed in Eosinophils
Sumika Toyama1,2, Okada Naoko, PhD1, Akio Matsuda, PhD1, Hirohisa Saito, MD, PhD.1, Susumu Nakae, PhD3, Hajime Karasuyama, MD, PhD2, and Kenji Matsumoto, MD, PhD1; 1Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan, 2Tokyo Medical and Dentistry Graduate School, Japan, Tokyo, Japan, 3Laboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. RATIONALE: Eosinophils play important roles in asthma pathogenesis, especially in airway remodeling. Fibroblast proliferation and production of extracellular matrix proteins, especially upon protease exposure, is an important feature of airway remodeling. However, we do not know if, or how, eosinophils are involved in that fibroblast activation. In the present study, we examined induction of eosinophil-specific proteases/proteinases by transcriptome analysis and tested their effects on human fibroblast. METHODS: Human eosinophils and other blood cells were purified from peripheral blood of mildly allergic donors, and protease/proteinase expression was measured using microarrays, qPCR and immunofluorescent staining. Eosinophils were stimulated with secretagogues, and PRSS33 concentrations in the supernatants and cell lysates were measured by ELISA. Cell surface expression of PRSS33 was determined by immunofluorescent staining and FACS. Recombinant PRSS33 protein (rPRSS33) was created by baculovirus construction, and mRNAs of extracellular matrix proteins in human primary nasal fibroblasts were measured by qPCR. RESULTS: Human eosinophils specifically and constitutively expressed mRNA for a novel protease, PRSS33 (serine protease 33), and the expression levels were not altered by eosinophil-activating cytokines. Immunofluorescent staining showed PRSS33 was co-localized with an eosinophil granule protein. PRSS33 protein was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but the cell surface expression was increased. Fibronectin and collagen mRNA expression were increased in human fibroblasts by rPRSS33 stimulation via PAR-2 activation. CONCLUSIONS: Eosinophils may activate fibroblast extracellular matrix protein synthesis through cell surface expression of PRSS33, which would explain their role in airway remodeling at least in part.
522
A novel biased antagonist of the eotaxin-CCR3 pathway in eosinophils
Milica M. Grozdanovic, PhD1, Lee K. Rousslang1, Hazem Abdelkarim, PhD1, Kimberly G. Laffey, PhD2, Ben Hitchinson, BSc1, Nadya Tarasova, PhD3, Vadim Gaponenko, PhD1, and Steven J. Ackerman, PhD1; 1University of Illinois at Chicago, Chicago, IL, 2Mayo Clinic, Rochester, MN, 3National Cancer Institute at Frederick, Frederick, MD. RATIONALE: The CCR3 signaling pathway is a key regulatory pathway for eosinophil tissue migration associated with allergic inflammation and asthma pathogenesis; however, none of the CCR3 antagonists developed to date have been approved for clinical use. A possible underlying reason for this failure is their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. We sought to develop a novel self-assembling peptide inhibitor (R321) with a biased mode of CCR3 antagonism. METHODS: Self-assembly of the R321 peptide into nanoparticles was analyzed by dynamic light scattering. Inhibitory activity onCCR3 signaling was assessed by determining IC50/IC90 values for eotaxininduced chemotaxis of CCR3+ cell lines and eosinophils. Mechanism of inhibition was determined by NMR on reductively methylated membranes and western blot analysis of ERK1/2 phosphorylation in CCR3+ cells. RESULTS: The R321 peptide self-assembles into monodisperse nanoparticles with R57.5 nm. IC50 values for eotaxin-(CCL11, CCL24,
CCL26) induced chemotaxis were in the low nanomolar range (210, 140, 100nM, respectively). R321inhibited only the early phase of ERK1/2 activation and not the late phase associated with b-arrestin recruitment and receptor internalization, promoting CCL11 and RANTES (CCL5)-induced CCR3 internalization and degradation. Binding of R321 to CCR3 did not cause displacement of CCL11, indicating a non-competitive allosteric inhibition. CONCLUSIONS: We evaluated the activity of a novel CCR3 peptide nanoparticle inhibitor with a potent inhibitory activity on CCR3-mediated chemotaxis. By demonstrating selective inhibition of only a subset of the CCR3 signaling cascade through a biased mode of antagonism, the peptide may hold significant therapeutic promise by eluding the formation of drug tolerance.
523
Label Free Identification of Peripheral Blood Eosinophils Using High-Throughput Imaging Flow Cytometry
Justyna Piasecka1, Holger Hennig2, Fabian J. Theis2, Paul Rees3,4, Huw D. Summers3, and Catherine A. Thornton1; 1Swansea University Medical School, Swansea, United Kingdom, 2Ingolstadter Landstr. 1, Helmholtz Zentrum Munchen, Neuherberg, Germany, 3Swansea University, College of Engineering, Swansea, United Kingdom, 4Imaging Platform at the Broad Institute of Harvard and MIT, Cambridge, MA. RATIONALE: Eosinophil degranulation patterns can provide insight into the contribution of eosinophils to disease. Current microscopy based approaches are labour intensive so limited for high throughput analysis in a clinical setting. Therefore, the aims of our work were to link different stimuli to human blood eosinophil degranulation patterns and progress to the development of a rapid, label-free method for monitoring blood eosinophil activation status. Such a development could provide a reliable strategy for early detection and monitoring of clinically relevant eosinophilia. METHODS: Peripheral blood was collected from healthy volunteers. Whole blood and isolated eosinophils were exposed to stimuli known to induce piecemeal degranulation - eotaxin and platelet activating factor (PAF). Changes to eosinophil morphology were recorded by conventional and imaging flow cytometry. Data were analysed using IDEAS software; statistical analysis was with Cell Profiler and MATLAB. RESULTS: Eosinophil autofluorescence and scatter signal intensity (relative to total cell population mean) from scatter, bright field and autofluorescence channels can be used to differentiate between activated and non-activated cells for both isolated eosinophils and within whole blood. These changes reflect differences in eosinophil degranulation patterns. CONCLUSIONS: A label-free, imaging flow cytometry-based approach can reveal different eosinophil degranulation patterns within whole human blood. This approach has the potential to provide identification of eosinophil phenotypes in a diagnostic setting. The goal is to create a diagnostic test that will provide currently unavailable insight and understanding to clinical practice for eosinophil identification and activation status using a fast, cheap, and reliable technique compatible with existing blood-based diagnostic services within hospitals.
SUNDAY
521
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
A Novel Protease, PRSS33 (serine protease 33), Is Specifically and Constitutively Expressed in Eosinophils
Sumika Toyama1,2, Okada Naoko, PhD1, Akio Matsuda, PhD1, Hirohisa Saito, MD, PhD.1, Susumu Nakae, PhD3, Hajime Karasuyama, MD, PhD2, and Kenji Matsumoto, MD, PhD1; 1Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan, 2Tokyo Medical and Dentistry Graduate School, Japan, Tokyo, Japan, 3Laboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. RATIONALE: Eosinophils play important roles in asthma pathogenesis, especially in airway remodeling. Fibroblast proliferation and production of extracellular matrix proteins, especially upon protease exposure, is an important feature of airway remodeling. However, we do not know if, or how, eosinophils are involved in that fibroblast activation. In the present study, we examined induction of eosinophil-specific proteases/proteinases by transcriptome analysis and tested their effects on human fibroblast. METHODS: Human eosinophils and other blood cells were purified from peripheral blood of mildly allergic donors, and protease/proteinase expression was measured using microarrays, qPCR and immunofluorescent staining. Eosinophils were stimulated with secretagogues, and PRSS33 concentrations in the supernatants and cell lysates were measured by ELISA. Cell surface expression of PRSS33 was determined by immunofluorescent staining and FACS. Recombinant PRSS33 protein (rPRSS33) was created by baculovirus construction, and mRNAs of extracellular matrix proteins in human primary nasal fibroblasts were measured by qPCR. RESULTS: Human eosinophils specifically and constitutively expressed mRNA for a novel protease, PRSS33 (serine protease 33), and the expression levels were not altered by eosinophil-activating cytokines. Immunofluorescent staining showed PRSS33 was co-localized with an eosinophil granule protein. PRSS33 protein was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but the cell surface expression was increased. Fibronectin and collagen mRNA expression were increased in human fibroblasts by rPRSS33 stimulation via PAR-2 activation. CONCLUSIONS: Eosinophils may activate fibroblast extracellular matrix protein synthesis through cell surface expression of PRSS33, which would explain their role in airway remodeling at least in part.
522
A novel biased antagonist of the eotaxin-CCR3 pathway in eosinophils
Milica M. Grozdanovic, PhD1, Lee K. Rousslang1, Hazem Abdelkarim, PhD1, Kimberly G. Laffey, PhD2, Ben Hitchinson, BSc1, Nadya Tarasova, PhD3, Vadim Gaponenko, PhD1, and Steven J. Ackerman, PhD1; 1University of Illinois at Chicago, Chicago, IL, 2Mayo Clinic, Rochester, MN, 3National Cancer Institute at Frederick, Frederick, MD. RATIONALE: The CCR3 signaling pathway is a key regulatory pathway for eosinophil tissue migration associated with allergic inflammation and asthma pathogenesis; however, none of the CCR3 antagonists developed to date have been approved for clinical use. A possible underlying reason for this failure is their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. We sought to develop a novel self-assembling peptide inhibitor (R321) with a biased mode of CCR3 antagonism. METHODS: Self-assembly of the R321 peptide into nanoparticles was analyzed by dynamic light scattering. Inhibitory activity onCCR3 signaling was assessed by determining IC50/IC90 values for eotaxininduced chemotaxis of CCR3+ cell lines and eosinophils. Mechanism of inhibition was determined by NMR on reductively methylated membranes and western blot analysis of ERK1/2 phosphorylation in CCR3+ cells. RESULTS: The R321 peptide self-assembles into monodisperse nanoparticles with R57.5 nm. IC50 values for eotaxin-(CCL11, CCL24,
CCL26) induced chemotaxis were in the low nanomolar range (210, 140, 100nM, respectively). R321inhibited only the early phase of ERK1/2 activation and not the late phase associated with b-arrestin recruitment and receptor internalization, promoting CCL11 and RANTES (CCL5)-induced CCR3 internalization and degradation. Binding of R321 to CCR3 did not cause displacement of CCL11, indicating a non-competitive allosteric inhibition. CONCLUSIONS: We evaluated the activity of a novel CCR3 peptide nanoparticle inhibitor with a potent inhibitory activity on CCR3-mediated chemotaxis. By demonstrating selective inhibition of only a subset of the CCR3 signaling cascade through a biased mode of antagonism, the peptide may hold significant therapeutic promise by eluding the formation of drug tolerance.
523
Label Free Identification of Peripheral Blood Eosinophils Using High-Throughput Imaging Flow Cytometry
Justyna Piasecka1, Holger Hennig2, Fabian J. Theis2, Paul Rees3,4, Huw D. Summers3, and Catherine A. Thornton1; 1Swansea University Medical School, Swansea, United Kingdom, 2Ingolstadter Landstr. 1, Helmholtz Zentrum Munchen, Neuherberg, Germany, 3Swansea University, College of Engineering, Swansea, United Kingdom, 4Imaging Platform at the Broad Institute of Harvard and MIT, Cambridge, MA. RATIONALE: Eosinophil degranulation patterns can provide insight into the contribution of eosinophils to disease. Current microscopy based approaches are labour intensive so limited for high throughput analysis in a clinical setting. Therefore, the aims of our work were to link different stimuli to human blood eosinophil degranulation patterns and progress to the development of a rapid, label-free method for monitoring blood eosinophil activation status. Such a development could provide a reliable strategy for early detection and monitoring of clinically relevant eosinophilia. METHODS: Peripheral blood was collected from healthy volunteers. Whole blood and isolated eosinophils were exposed to stimuli known to induce piecemeal degranulation - eotaxin and platelet activating factor (PAF). Changes to eosinophil morphology were recorded by conventional and imaging flow cytometry. Data were analysed using IDEAS software; statistical analysis was with Cell Profiler and MATLAB. RESULTS: Eosinophil autofluorescence and scatter signal intensity (relative to total cell population mean) from scatter, bright field and autofluorescence channels can be used to differentiate between activated and non-activated cells for both isolated eosinophils and within whole blood. These changes reflect differences in eosinophil degranulation patterns. CONCLUSIONS: A label-free, imaging flow cytometry-based approach can reveal different eosinophil degranulation patterns within whole human blood. This approach has the potential to provide identification of eosinophil phenotypes in a diagnostic setting. The goal is to create a diagnostic test that will provide currently unavailable insight and understanding to clinical practice for eosinophil identification and activation status using a fast, cheap, and reliable technique compatible with existing blood-based diagnostic services within hospitals.
SUNDAY
521